Chemical leads for the pharmaceutical industry are currently identified through rational design and/or mass screening. The recent introduction of high throughput, automated screening technologies has permitted evaluation of hundreds of thousands of individual test molecules against a large number of targets. However, the source, diversity and functionality of large chemical libraries still remains a limitation in identifying new leads. Compound libraries commonly used in mass screening consist of either a historical collection of synthesized compounds or natural product collections. Historical collections contain a limited number of diverse structures and represent only a small fraction of diversity possibilities. They also contain a limited number of biologically useful compounds. Natural products are limited by the structural complexity of the leads identified and the difficulty of reducing them to useful pharmaceutical agents (e.g., taxol).
Methods available for generating synthetic compound libraries differ considerably in the types and numbers of compounds prepared, and whether the compounds are obtained as single structurally defined entities or as large mixtures. New compound libraries have been obtained through rapid chemical and biological synthesis (Moos et al., Ann. Rep. Med. Chem. (1993) 28:315-324; Pavia et al., Bioorganic Medicinal Chem. Lett. (1993) 3:387-96; Gallop et al., J. Med. Chem., (1994) 37:1233-1251; Gordon et al, J. Med. Chem. (1994) 37:1385-1401). Peptide libraries containing hundreds to millions of small to medium size peptides have been made using “pin technology” representing a method that generates libraries of single compounds in a spatially-differentiated manner (Geysen et al., Proc. Nat. Acad. Sci. U.S.A. (1984), 81:3998-4002). The “spilt pool” method provides an alternative approach to preparing large mixtures of peptides and other classes of molecules (Furka et al., Abstr. 14th Int. Congr. Biochem., Prague, Czechoslovakia, Vol 5, pg 47. Abstr. 10th Intl. Symp. Med. Chem., Budapest, Hungary, (1988), pg 288; Houghten et al., Proc. Natl. Acad. Sci. U.S.A. (1985) 82:5131-35). Peptide libraries also have been produced by the “tea-bag” method in which small amounts of resins representing individual peptides are enclosed in porous polypropylene containers (Houghten et al., Nature (1991) 354:84-86). The bags are immersed in individual solutions of the appropriate activated amino acids while deprotections and washings are carried out by mixing all the bags together. The bags are then reseparated for subsequent coupling steps (the split-pool method). Removal of the peptides from the resins affords peptides in soluble form. It is possible to rapidly prepare a collection of libraries which represents, for example, all 64 million naturally-occurring hexapeptides and identify an optimal peptide ligand for any ligate of interest. Libraries of peptides also have been prepared on polymeric beads by the split-pool method and incubated with a tagged ligate. Ligates with bound peptides are identified by visual inspection, physically removed, and microsequenced (Lam et al., Nature (1991) 354:82-84). The approach also can incorporate cleavable linkers on each bead where, after exposure to cleaving reagent, the beads release a portion of their peptides into solution for biological assay and still retain sufficient peptide on the bead for microsequencing. The pin, split-pool, and tea-bag methods and libraries generated therefrom are limited to relatively small peptides amenable to this technology and the difficulty in identifying functional peptides of interest.
Peptide libraries also have been prepared in which an “identifier” tag is attached to a solid support material coincident with each monomer using a split-pool synthesis procedure. The structure of the molecule on any bead identified through screening is obtained by decoding the identifier tags. Numerous methods of tagging the beads have now been reported. These include the use of single stranded oligonucleotides which have the advantage of being used as identifying tags as well as allowing for enrichment through the use for PCR amplification (Brenner et al., Proc. Natl. Acad. Sci. U.S.A. (1992) 89:5381-5383; Nielsen et al., J. Am. Chem. Soc. (1993) 115:9812-9813; Needels et al., Proc. Natl. Acad. Sci. USA (1993) 90:10700-10704). The use of halocarbon derivatives which are released from the active beads through photolysis and sequenced using electron capture capillary chromatography has also been described (Gallop et al., Journal of Medicinal Chemistry, (1994) 37:1233-1251). While identifier tags aid screening of large peptide libraries, peptides are likely to have limited therapeutic applicability when modulation of receptor activity involved in a particular disorder require interaction with whole proteins, or protein complexes.
Phage libraries containing tens of millions of filamentous phage clones have been used as a biological source for generating peptide libraries, with each clone displaying a unique peptide sequence on the bacteriophage surface (Smith G. P., Science (1985) 228:1315-1317; Cwirla et al., Proc. Natl. Acad. Sci. USA (1990) 87:6378-6382; Devlin et al., Science (1990) 249:404-406). In this method, the phage genome contains the DNA sequence encoding for the peptide. The ligate of interest is used to affinity purify phage that display binding peptides, the phage propagated in E. coli, and the amino acid sequences of the peptides displayed on the phage are identified by sequencing the corresponding coding region of the viral DNA. Tens of millions of peptides can be rapidly surveyed for binding. Initial libraries of short peptides generally afford relatively weak ligands. Longer epitope regions and/or constrained epitopes also have been prepared. Phage technology also has effectively been applied to proteins and antibodies demonstrating that protein domains can fold properly on the surface of phage. A limitation of this method is that only naturally occurring amino acids can be used and little is known about the effect of the phage environment, as well as contaminants from cellular debris and phage.
Peptoid libraries have been created which represent a collection of peptides having N-substituted glycines as peptoid monomers (Zuckermann et al., J. Med. Chem. (1994) 37:2678-2685; Bunin et al., J. Am. Chem. Soc. (1992) 114:10997-10998; DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. (1993) 90:6909-6913; Bunin et al., Proc. Natl. Acad. Sci. USA (1993) 91:4708-4712; Hogan et al. WO 94/01102). Structures of the resulting compounds are unique, likely to display unique binding properties, and incorporate important functionalities of peptides in a novel backbone. The methods generate single structurally well defined molecules in a solution format after cleavage from a solid support. A disadvantage of this approach is the lack of correlating structure with function in screening the modified peptides, as well as limited therapeutic application when small peptides are insufficient to mimic activity of a protein or protein complex.
While each of the technologies described above afford a large number of compounds, the usefulness of these systems for the effective rapid discovery of drug candidates is limited since all of them result in the identification of relatively small peptide ligands. In most cases, small peptides are not suited as drugs due to in vivo instability and lack of oral absorption. Furthermore, conversion of a peptide chemical lead into a pharmaceutically useful, orally active, non-peptide drug candidate is more difficult than identifying the original peptide lead since no general solution yet exists for designing effective peptide mimics.
Another significant limitation of the various approaches described above are the size and complexity of the libraries, whether they are generated as single compounds (active compound identified by it's physical location) or mixtures (active compound identified by it's tag for encoded libraries or through deconvolution, where an active compound is identified by iterative synthesis and screening of mixtures). In addition, the construction of random synthetic, native, and phage libraries have proven useful but fall short of providing a more rational approach in development of compound libraries for the identification of a novel lead chemical structure. Accordingly, there exists a need to develop new libraries comprising functionally diverse compounds to improve the drug discovery process.